DNA Polymerases

Genetics
Copyright Genetics Society of America

DNA Polymerases

DNA polymerases are proteins that synthesize new DNA strands using preexisting DNA strands as templates. Before one cell divides to produce two cells, the DNA containing the genetic information in it must be duplicated for the new cell, in a process known as polymerization . In human cells, duplicating the DNA genome requires the polymerization of 2.91 billion nucleotides, the building blocks of DNA. In the bacterium Escherichia coli, the polymerization of 4.64 million nucleotides is necessary to duplicate the genome for the new cell. In all cells, the DNA polymerases are the protein catalysts that link together the nucleotide building blocks of the new DNA polymer in an accurate and timely process that occurs during replication .

The DNA polymerases are also required to repair the DNA of the genome. The genome's DNA can be damaged by highly reactive molecules that are either produced in the cell during normal metabolic processes or brought into the cell from external sources. The damage, if not repaired, could result in the production of mutations in the genome or possibly cell death. Several DNA repair processes occurring in the cell have been identified that preserve the integrity of the genome by removing the damaged nucleotides and resynthesizing DNA by the DNA polymerases.

The DNA Polymerase Mechanism

All DNA polymerases share a common mechanism for DNA chain synthesis. The polymerization of DNA occurs by the linkage of one nucleotide at a time to the end of a preexisting DNA chain. The sequence fluctuations of the nucleotides on the DNA template upon which the DNA polymerase is moving determines which nucleotide is added onto the end of the growing DNA chain. If a thymine (T) nucleotide is positioned in the DNA template, for example, then an adenine (A) is polymerized onto the DNA chain opposite the thymine in the DNA template. If a guanine (G) nucleotide is positioned in the template, a cytosine (C) is linked to the growing DNA chain opposite the guanine. This polymerization process results in the synthesis of a DNA chain that is complementary , rather than identical, to the template strand of DNA, and is sequenced according to the proper Watson-Crick nucleotide base pairing rules. During replication, both strands of the duplex DNA molecule serve as templates. The DNA strands are separated, and each of the DNA strands is copied by the DNA polymerases. This process results in two identical copies of the original duplex DNA molecule being produced for the two cells.

The DNA polymerase uses the nucleoside triphosphate form of the deoxynucleotides to build the DNA polymer. The monophosphate form of the deoxynucleotide is incorporated into the growing DNA chain, and a pyrophosphate molecule, a kind of salt, is released. The DNA polymerase can add nucleotides only to the 3′-OH end of the growing DNA chain (see above diagram).
Therefore, DNA polymerization occurs in only one direction. Some DNA polymerases are highly processive, polymerizing many nucleotides to the 3′ end of the DNA chain before falling off the DNA template. Other DNA polymerases are distributive in nature, incorporating just one nucleotide and then falling off the DNA template.

Occasionally, the DNA polymerase will incorrectly polymerize a nucleotide onto the growing DNA chain. Removal of this misinserted nucleotide must be performed by a "proofreading" exonuclease , which is a substance that removes nucleotides from the 3′ end of the DNA molecule. The combined actions of DNA polymerases and proofreading exonucleases improve the accuracy of DNA synthesis and thus minimize introduction of errors into the genome.

The Variety of DNA Polymerases

Like all proteins, the DNA polymerases are encoded in genes. The genes that encode the human DNA polymerases are contained in the genomic DNA at various positions on several different chromosomes. In February 2001 the first "working draft" sequence of the human genome was published. Analysis of this sequence shows us that there are perhaps as many as fifteen different DNA polymerase genes in the human genome. Each of these genes encodes a different DNA polymerase protein. However, biochemists have not yet isolated all of these enzymes. A similar analysis of the Escherichia coli genome shows us that there are five different DNA polymerase genes present in this bacterium. The multitude of DNA polymerases in human and bacterial cells indicates a specialized role for the different enzymes in various aspects of DNA replication and repair, many of which have yet to be identified.

The human DNA polymerase α is encoded in the POLA (polymerase alpha) gene located on the human X chromosome. The DNA polymerases δ and ε are encoded in the POLD1 (polymerase delta 1) and POLE1
(polymerase epsilon 1) genes, which are located on chromosomes 19 and 12, respectively. These three DNA polymerases are most frequently associated with replication of the human genome. The DNA polymerase β is encoded by the POLB (polymerase beta) gene on chromosome 8 and is involved in DNA repair.

The DNA polymerase γ is encoded by the POLG (polymerase gamma) gene on chromosome 15 and replicates the DNA of the mitochondria . In Escherichia coli the DNA polymerase I is the most active. This enzyme functions in the bacterial cell to repair DNA, while the DNA polymerase III is responsible for replicating the genome. There are several additional DNA polymerases in human and in bacterial cells of which the precise function is not known. Some of these enzymes might be necessary to replicate genomic DNA that has been damaged. The ability to replicate damaged DNA could lead to mutations introduced into the genome but would preserve the life of the cell.

The amino acid sequences of the DNA polymerase proteins can be deduced from the genetic code contained in the DNA polymerase genes. Based on the amino acid sequences of the DNA polymerases, these proteins have been classified into several families. Analysis of these sequences reveals a relatively diverse collection of proteins with some very important similarities in specific amino acid regions along the length of the protein.

The similarities in amino acid sequences in certain parts of the DNA polymerase proteins tell us that these regions of the protein have been conserved throughout evolution. These specific amino acids are those that are important in the catalytic function of DNA polymerization by these proteins. Some of the similar amino acids are necessary for binding metal atoms that are needed by the DNA polymerase to carry out the polymerization reaction. Other amino acid sequences allow the DNA polymerase to hold on to the DNA and the four different deoxynucleoside triphosphates as the enzyme polymerizes the new DNA chain. Some DNA polymerases have the necessary amino acid sequences to generate a 3′ proofreading exonuclease domain (region), allowing the DNA polymerase to remove mistakes, or proofread, as it builds the DNA polymer.

The DNA polymerases range in size from just over three hundred amino acids in length to more than two thousand amino acids in length. Three-dimensional studies of these enzymes have shown that the DNA polymerases have a common protein fold that resembles the shape of a "right hand" (see diagram). The "thumb," "fingers," and "palm" form a pocket along which the DNA can move. The DNA molecule interacts with specific amino acids
located in the "palm" region of the DNA polymerase, and the "thumb" clamps down on the DNA to hold it as the DNA chain-elongation reaction proceeds. The amino acids that are common in many of the DNA polymerases are found in the regions where the enzyme contacts the DNA molecule.

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